This invention relates to the area of herpes virology. More particularly, it relates to a new enzyme and the use of that enzyme as a target for anti-viral therapy.
Herpes viruses are large double stranded DNA viruses that are responsible for a number of human diseases including chicken pox, shingles, fever blisters, salivary gland virus disease, and infectious mononucleosis. The seven human herpes viruses that have been described thus far are HSV-1, HSV-2, cytomegalovirus (CMV), Epstein-Barr Virus (EBV), varicella zoster virus (VZV), HHV-6, and HHV-7.
Maturation of herpes virus particles is believed to occur through the formation of a procapsid structure, which acquires DNA and an envelope to become an infectious virion. A herpes virus group-common protein referred to as the assembly protein in CMV, and as p40, VP22a, NCP-3, and ICP35e in HSV-1, is an abundant constituent of the herpes virus procapsid. The assembly protein is phosphorylated and proteolytically processed from a precursor molecule. It is absent from the mature virion, although its fate is unknown. These characteristics of the assembly protein have suggested an analogy between it and the bacteriophage scaffolding protein, which is an essential component for phage assembly but is not found in mature virus particles (Gibson et al. (1991) J. Virol. 64:1241-1249).
The proteolytic processing of the assembly protein has been implicated as a critical step in the maturation of the virus. A temperature sensitive (ts) mutant that is unable to process the HSV assembly protein homolog (p40) is incapable of producing DNA-containing capsids or virions (Preston et al. (1983) J. Virol. 45:1056-1064). Maturational processing of the simian CMV (SCMV) Colburn assembly protein results in loss of its carboxy terminus. (Gibson, 1991, supra.)
Up until the present time the enzyme responsible for the proteolytic maturation of the assembly protein has not been identified. Further, there is a need in the art for new agents for therapeutic treatment of herpes viruses.
It is an object of the invention to provide a preparation of a proteinase encoded by a herpes virus.
It is another object of the invention to provide a substrate for cleavage by a herpes virus proteinase.
It is yet another object of the invention to provide a kit for measuring activity of a herpes virus proteinase.
It is still another object of the invention to provide a method for measuring activity of a herpes virus proteinase.
It is another object of the invention to provide a recombinant DNA molecule which encodes a herpes virus proteinase.
It is yet another object of the invention to provide an inhibitor of a herpes virus proteinase.
These and other objects of the invention are provided by one or more of the embodiments described below.
In one embodiment of the invention a preparation of the proteinase encoded by a herpes virus is provided, said preparation being free of a intact infectious herpes virus virion DNA.
In another embodiment of the invention substrates for cleavage by a herpes virus proteinase are provided. One substrate comprises a polypeptide containing the amino acid sequence:
aa1-aa2-Ala-aa3 (SEQ ID NO:28-30),
wherein aa1 is Val or Leu, aa2 is a polar amino acid, and aa3 is Ser, Val, or Asn, wherein the proteinase cleaves the substrate on the carboxy terminal side of the Ala residue. Another substrate comprises a polypeptide containing the amino acid sequence:
Tyr-aa4-aa5-Ala-aa6 (SEQ ID NO:32),
wherein aa4 is Val or Leu, aa5 is Lys or Gln and aa6 is Ser or Asn, and wherein the proteinase cleaves the substrate on the carboxy terminal side of the Ala residue.
In yet another embodiment of the invention a kit is provided for measuring activity of a herpes virus proteinase. The kit comprises a proteinase encoded by a herpes virus, and a substrate for cleavage by said proteinase. The substrate comprises a polypeptide containing the amino acid sequence:
aa1-aa2-Ala-aa3 or Tyr-aa4-aa5-Ala-aa6 (SEQ ID NO:28, 29, 30, or 32)
wherein aa1 is Val or Leu, aa12 is a polar amino acid, aa, is Ser, Val, or Asn, aa4 is Val or Leu, aa5 is Lys or Gin and aa6 is Ser or Asn, wherein the proteinase cleaves the substrate on the carboxy terminal side of the Ala residue, said kit being substantially free of intact infectious herpes virus.
In still another embodiment of the invention a method is provided for measuring activity of a herpes virus proteinase. The method comprises the steps of: contacting a proteinase encoded by a herpes virus with a substrate for cleavage by said proteinase, said substrate comprising a polypeptide containing the amino acid sequence:
aa1-aa2-Ala-aa3 or Tyr-aa4-aa5-Ala-aa6) (SEQ ID NO:28, 29, 30, or 32)
wherein aa1 is Val or Leu, aa2 is a polar amino acid, aa3 is Ser, Val, or Asn, aa4 is Val or Leu, aa5 is Lys or Gin and aa6 is Ser or Asn, wherein the proteinase cleaves the substrate on the carboxy terminal side of the Ala residue, said step of contacting occurring in the absence of an intact infectious herpes virus virion DNA; and monitoring cleavage of said substrate.
In another embodiment of the invention a recombinant DNA molecule is provided which encodes at least a portion of the herpes virus proteinase, said portion having the ability to cleave a herpes virus assembly protein precursor.
In yet another embodiment of the invention an inhibitor of a herpes virus proteinase is provided. The inhibitor comprises a derivative of the substrate of the herpes virus proteinase. The inhibitor may differ from the substrate in the scissile peptide bond which is carboxyl to the Ala residue.
These, and other embodiments of the invention which will be obvious to one skilled in the art from the disclosure, are described in more detail below. These embodiments provide the art with a promising target for specific anti-viral therapeutic agents, which can be administered to humans and other animals without also impairing normal cellular functions.